MENZELLA Hugo Gabriel
congresos y reuniones científicas
Lecture: Kosan polyketide engineering technology
Newcastle, UK
Congreso; 14th International Symposium on the Biology of Actinomycetes.; 2007
Type I polyketide synthase (PKS) genes consist of modules that encode the structures of 2-carbon units in their polyketide products. Alteration or replacement of PKS modules can lead to novel products, but techniques to effect changes have been extremely slow due to the lack of convenient tools to rapidly engineer the natural hosts or manipulate the wild type genes for heterologous expression. To overcome these limitations we have created a generic design of synthetic PKS genes where the DNA sequences are codon-optimized for expression in an E. coli strain engineered for polyketide production. In the redesigned sequences all the PKS components –extender modules, loading modules, thioesterases, intrapeptide linkers and doking domains- are flanked by a repeated set of unique restriction sites, allowing facile cassette assembly and interchange of PKS parts. To test feasibility, we synthesized a collection of PKS components comprising 50 extender modules, five loading modules, four thioesterases and 20 pairs of docking domains. Next, we created different systems to rapidly evaluate the compatibility of unrelated components for the assembly of hybrid PKSs. In one example, interaction between unrelated extender modules was assessed by creating ~300 mini PKSs. Remarkably, nearly half the combinations made the predicted polyketide in E. coli. In another example, a two plasmid system was created for the high-throughput screening of synthetic pairs of docking domains to facilitate interaction between PKS modules. Several pairs were found to be functional in multiple contexts and added to the PKS engineering toolbox. Overall, more than 4 million bp of novel PKS gene sequences were assembled to validate our approach to identify functional combinations of unrelated PKS components. In these experiments the results augur well for the ongoing development of molecular tools to design and produce novel polyketides.